Developmental progression continues during embryonic diapause in the roe deer.

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.

Prolonged Heat Stress during Winter Diapause Alters the Expression of Stress-Response Genes in Ostrinia nubilalis (Hbn.).

During diapause, a state of temporarily arrested development, insects require low winter temperatures to suppress their metabolism, conserve energy stores and acquire cold hardiness. A warmer winter could, thus, reduce diapause incidence and duration in many species, prematurely deplete their energy reserves and compromise post-diapause fitness. In this study, we investigated the combined effects of thermal stress and the diapause program on the expression of selected genes involved in antioxidant defense and heat shock response in the European corn borer Ostrinia nubilalis. By using qRT-PCR, it has been shown that response to chronic heat stress is characterized by raised mRNA levels of grx and trx, two important genes of the antioxidant defense system, as well as of hsp70 and, somewhat, of hsp90, two major heat shock response proteins. On the other hand, the expression of hsc70, hsp20.4 and hsp20.1 was discontinuous in the latter part of diapause, or was strongly controlled by the diapause program and refractory to heat stress, as was the case for mtn and fer, genes encoding two metal storage proteins crucial for metal ion homeostasis. This is the first time that the effects of high winter temperatures have been assessed on cold-hardy diapausing larvae and pupae of this important corn pest.

Significance of the clock gene period in photoperiodism in larval development and production of diapause eggs in the silkworm Bombyx mori.

Many insects living in seasonal environments sense seasonal changes from photoperiod and appropriately regulate their development and physiological activities. Genetic researches have indicated the importance of a circadian clock system in photoperiodic time-measurement for photoperiodic regulations. However, most previous studies have focused on the effects on a single photoperiodic phenotype, without elucidating whether the circadian clock is involved in the core photoperiodic mechanism or only in the production of one target phenotype, such as diapause. Here, we focused on two different phenotypes in a bivoltine Kosetsu strain of the silkworm Bombyx mori, namely, embryonic diapause and larval development, and examined their photoperiodic responses and relationship to the circadian clock gene period. Photoperiod during the larval stage clearly influenced the induction of embryonic diapause and duration of larval development in the Kosetsu strain; short-day exposure leaded to the production of diapause eggs and shortened the larval duration. Genetic knockout of period inhibited the short-day-induced embryonic diapause. Conversely, in the period-knockout silkworms, the larval duration was shortened, but the photoperiodic difference was maintained. In conclusion, our results indicate that the period gene is not causally involved in the photoperiodic response of larval development, while that is essential for the short-day-induced embryonic diapause.

Environmental and molecular regulation of diapause formation in a scyphozoan jellyfish.

Understanding the mechanisms underlying diapause formation is crucial for gaining insight into adaptive survival strategies across various species. In this study, we aimed to uncover the pivotal role of temperature and food availability in regulating diapausing podocyst formation in the jellyfish Aurelia coerulea. Furthermore, we explored the cellular and molecular basis of diapause formation using single-cell RNA sequencing. Our results showed cell-type-specific transcriptional landscapes during podocyst formation, which were underscored by the activation of specific transcription factors and signalling pathways. In addition, we found that the heat shock protein-coding genes HSC70 and HSP90a potentially act as hub genes that regulate podocyst formation. Finally, we mapped the single-cell atlas of diapausing podocysts and identified cell types involved in metabolism, environmental sensing, defence and development that may collectively contribute to the long-term survival and regulated excystment of diapausing podocysts. Taken together, the findings of this study provide novel insights into the molecular mechanisms that regulate diapause formation and contributes to a better understanding of adaptive survival strategies in a variety of ecological contexts.

Downregulation of a CT10 regulator of kinase (Crk) promotes the formation of diapause embryos in the brine shrimp Artemia.

To survive under harsh environments, embryonic development of Artemia was arrested at the gastrula stage and released as the diapause embryo. Cell cycle and metabolism were highly suppressed in this state of quiescence. However, cellular mechanisms underlying diapause remain largely unclear. In this study, we found that the expression level of a CT10 regulator of kinase-encoding gene (Ar-Crk) in diapause embryos was significantly lower than non-diapause embryos at the early embryogenetic stage of Artemia. Knockdown of Ar-Crk by RNA interference induced formation of diapause embryos, while the control group produced nauplii. Western blot analysis and metabolic assays revealed that the diapause embryos produced by Ar-Crk-knocked-down Artemia had similar characteristics of diapause markers, arrested cell cycle, and suppressed metabolism with those diapause embryos produced by natural oviparous Artemia. Transcriptomic analysis of Artemia embryos revealed knockdown of Ar-Crk induced downregulation of the aurora kinase A (AURKA) signaling pathway, as well as energetic and biomolecular metabolisms. Taken together, we proposed that Ar-Crk is a crucial factor in determining the process of diapause in Artemia. Our results provide insight into the functions of Crk in fundamental regulations such as cellular quiescence.

Semi-field and surveillance data define the natural diapause timeline for Culex pipiens across the United States.

Reproductive diapause serves as biological mechanism for many insects, including the mosquito Culex pipiens, to overwinter in temperate climates. While Cx. pipiens diapause has been well-studied in the laboratory, the timing and environmental signals that promote diapause under natural conditions are less understood. In this study, we examine laboratory, semi-field, and mosquito surveillance data to define the approximate timeline and seasonal conditions that contribute to Cx. pipiens diapause across the United States. While confirming integral roles of temperature and photoperiod in diapause induction, we also demonstrate the influence of latitude, elevation, and mosquito population genetics in shaping Cx. pipiens diapause incidence across the country. Coinciding with the cessation of WNV activity, these data can have important implications for mosquito control, where targeted efforts prior to diapause induction can decrease mosquito populations and WNV overwintering to reduce mosquito-borne disease incidence the following season.

Time-Series Transcriptomic Analysis Reveals the Molecular Profiles of Diapause Termination Induced by Long Photoperiods and High Temperature in Chilo suppressalis.

Survival and adaptation to seasonal changes are challenging for insects. Many temperate insects such as the rice stem borer (Chilo suppressalis) overcome the adverse situation by entering diapause, wherein development changes dynamically occur and metabolic activity is suppressed. The photoperiod and temperature act as major environmental stimuli of diapause. However, the physiological and molecular mechanisms that interpret the ecologically relevant environmental cues in ontogenetic development during diapause termination are poorly understood. Here, we used genome-wide high-throughput RNA-sequencing to examine the patterns of gene expression during diapause termination in C. suppressalis. Major shifts in biological processes and pathways including metabolism, environmental information transmission, and endocrine signalling were observed across diapause termination based on over-representation analysis, short time-series expression miner, and gene set enrichment analysis. Many new pathways were identified in diapause termination including circadian rhythm, MAPK signalling, Wnt signalling, and Ras signalling, together with previously reported pathways including ecdysteroid, juvenile hormone, and insulin/insulin-like signalling. Our results show that convergent biological processes and molecular pathways of diapause termination were shared across different insect species and provided a comprehensive roadmap to better understand diapause termination in C. suppressalis.

Nutrient sensing pathways regulating adult reproductive diapause in C. elegans.

Genetic and environmental manipulations, such as dietary restriction, can improve both health span and lifespan in a wide range of organisms, including humans. Changes in nutrient intake trigger often overlapping metabolic pathways that can generate distinct or even opposite outputs depending on several factors, such as when dietary restriction occurs in the lifecycle of the organism or the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis elegans is a dietary restriction model that is associated with lengthened lifespan and reproductive potential. To explore the metabolic pathways regulating ARD in greater depth, we performed a candidate-based genetic screen analyzing select nutrient-sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three phases of ARD (initiation, maintenance, and recovery), we found that ARD initiation is regulated by fatty acid metabolism, sirtuins, AMPK, and the O-linked N-acetyl glucosamine (O-GlcNAc) pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in our screen, we found that ARD recovery was modulated by energy sensing, stress response, insulin-like signaling, and the TOR pathway. Further investigation of downstream targets of NHR-49 suggest the transcription factor influences ARD initiation through the fatty acid ß-oxidation pathway. Consistent with these findings, our analysis revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings identify conserved genetic pathways required for ARD entry and recovery and uncover genetic interactions that provide insight into the role of OGT and OGA.

YTHDF3 Is Involved in the Diapause Process of Bivoltine Bombyx mori Strains by Regulating the Expression of Cyp307a1 and Cyp18a1 Genes in the Ecdysone Synthesis Pathway.

The variable diapause features of bivoltine silkworm (Bombyx mori) strains regulated by environmental signals in the embryonic stage are closely related to epigenetics. Previously, we showed that the expression of YTHDF3 is significantly different in the pupae of the bivoltine silkworm Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, nondiapause egg producer), indicating that the expression of diapause-associated genes is regulated by the m6A modification level. However, how YTHDF3 regulates the expression of diapause-related genes remains unclear. In this study, we observed that the knockdown of B. mori YTHDF3 resulted in delayed embryo development, while the overexpression of YTHDF3 resulted in the transformation of nondiapause-destined eggs into a mixture of diapause and nondiapause eggs. Further studies showed that YTHDF3, as a reading protein, can recognize the m6A site of Cyp307a1 and Cyp18a1 genes in the ecdysone synthesis pathway (ESP), and the overexpression of YTHDF3 affects the diapause traits of the silkworm by decreasing the stabilities of mRNAs of Cyp307a1 and Cyp18a1 and inhibiting their translation. The above results demonstrate that m6A modification mediates YTHDF3 to affect the expression levels of its target genes, Cyp307a1 and Cyp18a1, in the ESP to regulate diapause in bivoltine B. mori. This is the first report of the m6A methylation regulation mechanism in diapause in B. mori and provides new experimental data for clarifying the diapause regulation network.

Cathepsin L Contributes to Reproductive Diapause by Regulating Lipid Storage and Survival of Coccinella septempunctata (Linnaeus).

Cathepsin L protease, which belongs to the papain-like cysteine proteases family, is an important player in many physiological and pathological processes. However, little was known about the role of cathepsin L in ladybird beetles (Coccinella septempuctata Linnaeus) during diapause. Here, we analyzed the characteristics of cathepsin L (CsCatL) in the females of C. septempunctata and its role during the diapause of the ladybeetle. CsCatL was cloned and identified from beetle specimens by rapid amplification of cDNA-ends (RACE). The cDNA sequence of CsCatL was 971 bp in length, including an 843 bp open reading frame encoding a protein of 280 amino acids. It was identified as the cathepsin L group by phylogenetic analysis. Knockdown of CsCatL by RNA interference led to decreased expression levels of fatty acid synthase 2 (fas 2) genes and suppressed lipid accumulation. Furthermore, silencing the CsCatL gene distinctly reduced diapause-related features and the survival of female C. spetempunctata under diapause-inducing conditions. The results suggested that the CsCatL gene was involved in fatty acid biosynthesis and played a crucial role in the survival of adult C. septempunctata during the diapause preparation stage.

Extensive transcriptomic profiling of pupal diapause in a butterfly reveals a dynamic phenotype.

Diapause is a common adaptation for overwintering in insects that is characterized by arrested development and increased tolerance to stress and cold. While the expression of specific candidate genes during diapause have been investigated, there is no general understanding of the dynamics of the transcriptional landscape as a whole during the extended diapause phenotype. Such a detailed temporal insight is important as diapause is a vital aspect of life cycle timing. Here, we performed a time-course experiment using RNA-Seq on the head and abdomen in the butterfly Pieris napi. In both body parts, comparing diapausing and nondiapausing siblings, differentially expressed genes are detected from the first day of pupal development and onwards, varying dramatically across these formative stages. During diapause there are strong gene expression dynamics present, revealing a preprogrammed transcriptional landscape that is active during the winter. Different biological processes appear to be active in the two body parts. Finally, adults emerging from either the direct or diapause pathways do not show large transcriptomic differences, suggesting the adult phenotype is strongly canalized.

Characterization and Developmental Expression Patterns of Four Hexamerin Genes in the Bumble Bee, Bombus terrestris (Hymenoptera: Apidae).

Hexamerins are members of the hemocyanin superfamily and play essential roles in providing amino acids and energy for the nonfeeding stages of insects. In this study, we cloned and analyzed the expression patterns of four hexamerin genes (hex 70a, hex 70b, hex 70c, and hex 110) at different worker development stages and queen diapause statuses in the bumble bee, Bombus terrestris. The results of this study showed that hex 110 has the longest open reading frame (ORF; 3,297 bp) compared to the ORFs of hex 70a (2,034 bp), hex 70b (2,067 bp), and hex 70c (2,055 bp). The putative translation product of Hex 70a, Hex 70b, Hex70c, and Hex 110 has 677, 688, 684, and 1,098aa with predicted molecular mass of 81.13, 79.69, 81.58, and 119 kDa. In the development stages of workers, the expression levels of hex 70a, hex 70b, and hex 70c increased gradually from the larval stage and exhibited high expression levels at the pink eyed and brown eyed pupae stage, whereas hex 110 exhibited the highest expression level at the larval period. Four hexamerin genes were highly expressed at the prediapause status of queen (P < 0.05), and compared to the eclosion queen, the lowest upregulation was 3.7-fold, and the highest upregulation was 1,742-fold. The expression levels of hex 70b, hex 70c, and hex 110 at diapause were significantly higher than those at postdiapause (P < 0.05). In conclusion, hexamerins may play important roles in queen diapause and metamorphosis of larval and pupal stages.

Production of cloned transgenic silkworms by breeding non-diapausing parthenogenetic strains.

Clonal transgenic silkworms are useful for the functional analysis of insect genes and for the production of recombinant proteins. Such silkworms have previously been created using an existing ameiotic parthenogenetic strain. However, the process was labor intensive, and the efficiency of producing transgenic silkworms was very low. To overcome this issue, we developed a more convenient and efficient method by breeding non-diapausing parthenogenetic strains. The strains produced non-diapausing eggs only when the embryogenesis of the parent eggs was performed at low temperatures, which could then be used for injecting vector plasmids. This demonstrated that transgenic silkworms could be produced with greater ease and efficiency. To breed the strains, we crossed the existing parthenogenetic strains with bivoltine strains and made F1 and F2 from each cross. Then we selected the silkworms whose eggs have a high ability of parthenogenesis and became non-diapausing. We also demonstrated that the germplasm could be cryopreserved in liquid nitrogen. Thus, this method increases the efficiency and ease of using genetically engineered silkworms to analyze gene function and produce recombinant proteins, potentially impacting various industries.

Post-diapause transcriptomic restarts: insight from a high-latitude copepod.

BACKGROUND: Diapause is a seasonal dormancy that allows organisms to survive unfavorable conditions and optimizes the timing of reproduction and growth. Emergence from diapause reverses the state of arrested development and metabolic suppression returning the organism to an active state. The physiological mechanisms that regulate the transition from diapause to post-diapause are still unknown. In this study, this transition has been characterized for the sub-arctic calanoid copepod Neocalanus flemingeri, a key crustacean zooplankter that supports the highly productive North Pacific fisheries. Transcriptional profiling of females, determined over a two-week time series starting with diapausing females collected from > 400 m depth, characterized the molecular mechanisms that regulate the post-diapause trajectory. RESULTS: A complex set of transitions in relative gene expression defined the transcriptomic changes from diapause to post-diapause. Despite low temperatures (5-6 °C), the switch from a "diapause" to a "post-diapause" transcriptional profile occurred within 12 h of the termination stimulus. Transcriptional changes signaling the end of diapause were activated within one-hour post collection and included the up-regulation of genes involved in the 20E cascade pathway, the TCA cycle and RNA metabolism in combination with the down-regulation of genes associated with chromatin silencing. By 12 h, females exhibited a post-diapause phenotype characterized by the up-regulation of genes involved in cell division, cell differentiation and multiple developmental processes. By seven days post collection, the reproductive program was fully activated as indicated by up-regulation of genes involved in oogenesis and energy metabolism, processes that were enriched among the differentially expressed genes. CONCLUSIONS: The analysis revealed a finely structured, precisely orchestrated sequence of transcriptional changes that led to rapid changes in the activation of biological processes paving the way to the successful completion of the reproductive program. Our findings lead to new hypotheses related to potentially universal mechanisms that terminate diapause before an organism can resume its developmental program.

Steroid hormone pathways coordinate developmental diapause and olfactory remodeling in Pristionchus pacificus.

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate the development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole hydroxysteroid dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal-associated neurons (CANs) and excretory cells whose homologous cells in Caenorhabditis elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.

Diapause vs. reproductive programs: transcriptional phenotypes in a keystone copepod.

Many arthropods undergo a seasonal dormancy termed "diapause" to optimize timing of reproduction in highly seasonal environments. In the North Atlantic, the copepod Calanus finmarchicus completes one to three generations annually with some individuals maturing into adults, while others interrupt their development to enter diapause. It is unknown which, why and when individuals enter the diapause program. Transcriptomic data from copepods on known programs were analyzed using dimensionality reduction of gene expression and functional analyses to identify program-specific genes and biological processes. These analyses elucidated physiological differences and established protocols that distinguish between programs. Differences in gene expression were associated with maturation of individuals on the reproductive program, while those on the diapause program showed little change over time. Only two of six filters effectively separated copepods by developmental program. The first one included all genes annotated to RNA metabolism and this was confirmed using differential gene expression analysis. The second filter identified 54 differentially expressed genes that were consistently up-regulated in individuals on the diapause program in comparison with those on the reproductive program. Annotated to oogenesis, RNA metabolism and fatty acid biosynthesis, these genes are both indicators for diapause preparation and good candidates for functional studies.

Elucidating evolutionarily conserved mechanisms of diapause regulation using an in silico approach.

Embryonic diapause is an enigmatic phenomenon that appears in diverse species. Although regulatory mechanisms have been established, there is much to be discovered. Herein, we have made the first comprehensive attempt to elucidate diapause regulatory mechanisms using a computational approach. We found transcription factors unique to promoters of genes in diapause species. From pathway analysis and STRING PPI networks, the signaling pathways regulated by these unique transcription factors were identified. The pathways were then consolidated into a model to combine various known mechanisms of diapause regulation. This work also highlighted certain transcription factors that may act as 'master transcription factors' to regulate the phenomenon. Promoter analysis further suggested evidence for independent evolution for some of regulatory elements involved in diapause.

Hormonal Regulation of Reproductive Diapause That Occurs in the Year-Round Mass Rearing of Bombus terrestris Queens.

Adult reproductive diapause is an adaptive strategy under adverse environments for insects and other arthropod species, including bumblebees, which enables queens to survive through a harsh winter and then build new colonies in the following spring. Little research has been done on the molecular regulatory mechanism of reproductive diapause in Bombus terrestris, which is an important pollinator of wild plants and crops. Our previous research identified the conditions that induced reproductive diapause during the year-round mass rearing of B. terrestris. Here, we performed combined transcriptomics and proteomics analyses of reproductive diapause in B. terrestris during and after diapause at three different ecophysiological phases, diapause, postdiapause, and founder postdiapause. The analyses showed that differentially expressed proteins/genes acted in the citrate cycle, insect hormone biosynthesis, insulin and mTOR signaling pathway. To further understand the mechanisms that regulated the reproductive diapause, genes involved in the regulation of JH synthesis, insulin/TOR signal pathway were determined. The BtRheb, BtTOR, BtVg, and BtJHAMT had lower expression levels in diapause queens. The JH III titer levels and the activities of the metabolic enzymes were significantly up-regulated in postdiapause queens. Also, after the microinjection of insulin-like peptides (ILPs) and JH analogue (JHA), hormones, cold-tolerance metabolites, metabolic enzymes, and reproduction showed significant changes. Together with results from other related research, a model of the regulation of reproductive diapause during the year-round mass rearing of B. terrestris was proposed. This study contributes to a comprehensive insight into the molecular regulatory mechanism of reproductive diapause in eusocial insects.

Steroid hormone ecdysone deficiency stimulates preparation for photoperiodic reproductive diapause.

Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.

Transcriptomic analysis elucidates the molecular processes associated with hydrogen peroxide-induced diapause termination in Artemia-encysted embryos.

Treatment with hydrogen peroxide (H2O2) raises the hatching rate through the development and diapause termination of Artemia cysts. To comprehend the upstream genetic regulation of diapause termination activated by exterior H2O2 elements, an Illumina RNA-seq analysis was performed to recognize and assess comparative transcript amounts to explore the genetic regulation of H2O2 in starting the diapause termination of cysts in Artemia salina. We examined three groupings treated with no H2O2 (control), 180 µM H2O2 (low) and 1800 µM H2O2 (high). The results showed a total of 114,057 unigenes were identified, 41.22% of which were functionally annotated in at least one particular database. When compared to control group, 34 and 98 differentially expressed genes (DEGs) were upregulated in 180 µM and 1800 µM H2O2 treatments, respectively. On the other hand, 162 and 30 DEGs were downregulated in the 180 µM and 1800 µM H2O2 treatments, respectively. Cluster analysis of DEGs demonstrated significant patterns among these types of 3 groups. GO and KEGG enrichment analysis showed the DEGs involved in the regulation of blood coagulation (GO: 0030193; GO: 0050818), regulation of wound healing (GO:0061041), regulation of hemostasis (GO: 1900046), antigen processing and presentation (KO04612), the Hippo signaling pathway (KO04391), as well as the MAPK signaling pathway (KO04010). This research helped to define the diapause-related transcriptomes of Artemia cysts using RNA-seq technology, which might fill up a gap in the prevailing body of knowledge.